The long-term objective of this proposal is to explore how the nuclear receptors of thyroid hormones regulate the expression of specific cellular genes. There are two major types of thyroid hormone receptors, TRalpha and TRbeta. These receptors function as ligand-inducible transcription factors. Hormone-free TRs actively repress transcription of genes bearing TR-response elements. Ligand binding to the receptor triggers the release of transcriptional silencing an leads to the activation of target gene expression. The specific aims of this proposal are to: (1) Define the components that are necessary to reconstitute TRbeta-directed silencing of target gene transcription ina purified cell-free system. TRbeta mediated silencing of a target gene and its hormone-induced reversal have been reconstituted in cell-free HeLa nuclear extracts. Recent experiments suggests the existence of a regulatory molecular that interacts with the C- terminal ligand binding domain (LBD) of TRbeta and is essential for efficient receptor-mediated silencing. This soluble corepressor activity will be purified from HeLa nuclear extracts and its biochemical and functional properties will be analyzed. A well-defined cell-free system will be reconstituted by combining highly purified receptor, corepressor, RNA polymerase II and other basal transcription factors. (2) Map the functional domains of TRbeta involved in transcriptional silencing. To locate precisely the region(s) that constitute this function, extensive mutagenesis will be performed within the 300 amino acid long LBD of TRbeta. The mutant proteins will be expressed in E. coli and purified by affinity chromatography. The silencing activities of these mutants will be analyzed in a cell-free transcription system. Natural mutations in hTRbeta gene have been identified in human patients with generalized resistance to thyroid hormone (GRTH). Many of these mutants are impaired in ligand binding activity and therefore may function as constitutive silencers of genes regulated by wild-type TR or related nuclear receptors. Known GRTH mutants will be expressed in E. coli and their silencing activities will be analyzed. These experiments will allow us to determine whether constitutive silencing by these mutant receptors contribute to the perturbation of wild-type receptor activity in the heterozygous kindreds with GRTH syndromes. (3) Define the mechanisms by which TRbeta interacts with the components of the RNA polymerase II transcription machinery to silence target gene transcription. Recent studies indicate that unliganded TRbeta arrests the assembly of a functional preinitiation complex at the target promoter. SA major focus of this proposal is to identify the assembling complex (es) that are targets of inhibition by TRbeta during the stepwise generation of the preinitiation complex. The interactions of the receptor with these core complexes and modulatory effects of thyroid hormone on these interactions will be investigated by protein-protein interaction assays. The functional consequence of these interactions will be monitored by cell-free transcription experiments. The proposed research will help us to understand the fundamental mechanisms that determine the gene regulatory activity of TR in normal cellular physiology and in human disease states such as GRTH.